Elevated nuclear TDP-43 induces constitutive exon skipping

Background Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate in vitro and in vivo model systems. Methods We analyzed RNA-seq datasets from mouse and human neurons overexpressing TDP-43 to explore species specific splicing patterns. We explored the dynamics between TDP-43 levels and exon repression in vitro. Furthermore we analyzed human brain samples and publicly available RNA datasets to explore the relationship between exon repression and disease. Results Our study shows that excessive levels of nuclear TDP-43 protein lead to constitutive exon skipping that is largely species-specific. Furthermore, while aberrant exon skipping is detected in some human brains, it is not correlated with disease, unlike the incorporation of cryptic exons that occurs after loss of TDP-43. Conclusions Our findings emphasize the need for caution in interpreting TDP-43 overexpression data and stress the importance of controlling for exon skipping when generating models of TDP-43 proteinopathy. Supplementary Information The online version contains supplementary material available at 10.1186/s13024-024-00732-w.

Alternative strategies to model TDP-43 proteinopathy have been developed, involving the use of adenoassociated virus (AAV)-mediated delivery of TDP-43 [49] and the generation of transgenic TDP-43 models that feature mutated nuclear localization signals (NLS), such as rNLS8 [50][51][52][53].The use of AAV delivery bypasses development-specific effects and enables delivery to specific cell types in adult mice, while the animal models that express NLS-deficient TDP-43 enable the modelling of cytoplasmic toxicity [54] that are not recapitulated by wildtype overexpression [37].These studies indicate a potential unified mechanism of neurodegeneration in which microglia exert a neuroprotective role by phagocytically clearing pathological TDP-43 [49][50][51], whereas impaired microglial function could worsen neuronal damage and motor impairments.However, it is not known whether the molecular mechanisms that produce the pathological changes in these overexpression models are equivalent to those that occur in human disease.Fully characterizing these mechanisms will be crucial to accurately interpret TDP-43 overexpression models and their relevance to human disease.
Prior work in transgenic mouse models has demonstrated that increased expression of wild-type or mutant TDP-43 in mice caused widespread splicing changes, with differing effects depending on specific exons and TDP-43 variants [55].While elevated wild-type TDP-43 increased exon skipping for many exons, mutant TDP-43 showed both loss-of-function (reduced exon skipping) and gain-of-function (skipping of new exons) effects in an exon-dependent manner that also depended on levels of transgene expression.Further studies used mouse mutants carrying point mutations in endogenous Tardbp to identify a novel class of exons, termed "skiptic exons", that are skipped due to gain-of-function effects from these Tardbp point mutations [56].These skiptic exons show high conservation across species and 2 out of 7 skiptic events were validated in fibroblasts from ALS patients with disease-causing TARDBP mutations.Although TDP-43 RNA binding was proposed as the likely mediator of splicing gain-of-function, the authors also identified that various point mutations are able to disrupt autoregulation and lead to increased mRNA levels of Tardbp, as was reported in a parallel study [57].These findings highlight the nuanced impact of TDP-43 mutations and expression levels on alternative splicing regulation.
In this work, we demonstrate that excessive levels of nuclear TDP-43 protein, mutant or wildtype, leads to the repression of constitutive exons that are normally incorporated into mRNAs.Sufficiently high levels of TDP-43 overexpression can overwhelm autoregulation, even when nuclear localization-deficient isoforms of TDP-43 [50,52,58] are introduced.TDP-43 preferentially binds to long repetitive UG repeats [16,59], but with higher concentrations in the nucleus, we theorize that TDP-43 can bind to shorter, less optimal UG-containing motifs present at these constitutive exons.
Interestingly, the constitutive exons repressed by excessive TDP-43 are mostly divergent between mouse and human neurons.We also find that, while aberrant constitutive exon skipping can be detected in some human brain samples, constitutive exon skipping does not correlate with disease.By contrast, TDP-43 dependent cryptic exons are found only in human disease tissues or biofluids.Our findings imply that constitutive exon skipping is associated with TDP-43 overexpression, but its direct link to neurodegeneration requires further investigation in vivo.The consequences of skiptic splicing should be considered when developing models of TDP-43 proteinopathy to ensure that these models accurately reflect disease mechanisms.

Results
Transgenic models of TDP-43 overexpression exhibit a dose-dependent toxicity [31,[60][61][62], but do not recapitulate the nuclear clearance and cytoplasmic aggregation that are hallmarks of TDP-43 proteinopathy.TDP-43 overexpression models also do not exhibit any disruptions in cryptic exon repression, as found in models of TDP-43 loss-of-function [15].These findings suggest that increasing TDP-43 protein to a degree that significantly exceeds physiological levels introduces gain-of-function toxicity that may not be applicable to the pathogenesis of human disease.Therefore, to determine whether slight elevations in TDP-43 levels could produce better models of disease, we generated transgenic mice that express wildtype human TDP-43 (TDP-43 WT ) or human TDP-43 carrying the G298S mutation (TDP-43 G298S ) that is associated with familial ALS [63], under the control of the Thy1.2 promoter (Fig. 1A).Immunoblotting analysis indicates that total TDP-43 protein levels in these transgenic models was approximately 50% higher than in nontransgenic controls (Fig. 1B).Indeed, transgenic animals developed a progressive motor deficit that began with hind-limb clench and hemiparesis, eventually leading to end-stage paralysis.Behavioral testing showed marked reduction on hanging time in transgenic lines compared ) were expressed under the weak Thy1.2 promoter in mice.(B) TDP-43 levels in the spinal cord and cortex of transgenic mice were compared to control mice using immunoblotting.In both transgenic lines, TDP-43 protein levels were elevated at approximately 1.5x and 1.3x higher in the spinal cord and cortex, respectively (* p < 0,05, ***p < 0.001).(C) We measured the hanging time of transgenic mice compared to their littermate controls and found a reduction associated with age, indicative of a motor neuron deficit.(D) Both transgenic lines had shorter survival times compared to non-transgenic (NT) controls (NT vs. WT: p = 0.0005, NT vs. G298S: p = 0.0035), but no differences were found between the two transgenic lines (WT vs. G298S: p = 0.1260).(E-G) RNA-Seq analysis on isolated mouse spinal cords revealed several examples of exon skipping (arrows) in both transgenic lines.(H) We further validated these findings by transfecting mouse N2a cells with human TDP-43 and performing RT-PCR to test whether TDP-43 expression alone was sufficient to induce exon skipping with littermate controls (Fig. 1C) and animal weights for both transgenic mouse lines markedly decreased over time (Supplementary Fig. 1).The Kaplan-Meier survival curve indicated that lifespans of individuals from both transgenic lines were significantly shorter than their littermate controls (Fig. 1D).Pathological examination showed that TDP-43 overexpression was confined to the nucleus of neurons from the cortex and spinal cord, without any cytoplasmic inclusions in either transgenic line.Furthermore, histological analysis revealed reduction in axonal diameter, neuromuscular denervation and muscle degeneration in both transgenic lines, reflecting the severity of their condition (Supplementary Fig. 1).In summary, both TDP-43 WT and TDP-43 G298S mouse lines exhibited similar phenotypes of mild motor deficits that appeared to be mutation-independent.
We next hypothesized that the observed phenotypes could be explained by changes in gene expression or alternative splicing due to the relatively low levels of TDP-43 overexpression.To profile these transcriptomic changes, we surgically isolated the ventral horn of the spinal cord and conducted bulk RNA sequencing (RNA-Seq) on the TDP-43 WT and TDP-43 G298S lines, as well as non-transgenic controls.TDP-43 has been welldescribed as a splicing repressor and loss of TDP-43 function leads to the inclusion of nonconserved cryptic exons.Interestingly, analysis of transgenic RNA-Seq data revealed that overexpression of both TDP-43 WT and TDP-43 G298S leads to the skipping of constitutive conserved exons (Fig. 1E-G).To verify that these splicing events were indeed due to TDP-43 overexpression and not due to the secondary effects of neuronal degeneration, we overexpressed TDP-43 in mouse N2a cells and performed a RT-PCR to detect the same targets identified from TDP-43 WT and TDP-43 G298S transgenic RNA-Seq data.As predicted, we observed prominent constitutive exon skipping only following TDP-43 overexpression (Fig. 1H).Together, these data indicate that even mild overexpression of TDP-43 (50% increase over controls) is sufficient to induce aberrant exon skipping and that exon skipping could be mediating the dose-dependent toxicity of TDP-43 overexpression.
Given the evolutionary conservation of the coding exons skipped by TDP-43 overexpression in mice, we hypothesized that syntenic exons in the human genome might also be skipped by TDP-43 overexpression in human cells.To profile exon skipping, we transduced human i3Neurons [64] with lentivirus expressing TDP-43 and examined the splicing patterns of the equivalent exons observed to be skipped in mice (Fig. 2A).Unexpectedly, a large majority of skipped exons in mice were not skipped in human cells following TDP-43 overexpression, despite a ∼ 1.6x fold increase in TDP-43 protein (Supplementary Fig. 2).To understand these differences, we expanded our RNA-Seq analysis on transduced i3Neurons to include all splicing events and found that exon skipping still occurred across multiple genes (78 skipped exons, Supplementary Fig. 3) but at sites entirely different from those found in mice (Supplementary Table 1).We only found two exceptions in DDI2 and SLC6A6 where skipping occurred for equivalent mouse and human exons (Supplementary Fig. 3).Importantly, SLC6A6 has already been validated as a skiptic event in the TDP-43 M323K mouse line and human fibroblasts derived from ALS patients carrying TARDBP pathogenic mutations [56].The genes affected by exon skipping regulate a variety of cellular pathways including those associated with intellectual disability, synaptic activity, and mitochondrial proteins (Fig. 2B-C).Three of the clearest splicing repression events (∼ 90% reduction) include exons in HYOU1, NUP93, and XPNPEP1, where the corresponding exon in mice remains constitutively spliced (Fig. 2D-F).We confirmed that these human skipped exons we evolutionarily conserved (Supplementary Fig. 4) and further validated the RNA-Seq results by RT-PCR analysis (Fig. 2G-H).UG repeats serve as the consensus binding site for TDP-43.An analysis of exons repressed by TDP-43 overexpression reveals the presence of short UG motifs that may be responsible for speciesspecific exon skipping, but these UG motifs are not as long as UG repeats associated with cryptic exons (Fig. 2I, Supplementary Fig. 5) [15,16,59,65].This suggests that smaller UG repeats may be available for TDP-43 to bind when protein concentrations of nuclear TDP-43 exceed a certain threshold, as indicated by a limited number of eCLIP motifs adjacent to skipped exons (Supplementary Fig. 6).Overall, our data indicate that while TDP-43 overexpression leads to aberrant exon skipping across the transcriptome, the exons that are skipped appear to be species-specific and generally exhibit shorter UG repeats than cryptic exons (Fig. 3A).
(See figure on previous page.)Fig. 2 TDP-43 overexpression induces exon repression in humans.(A) Human i3Neurons were transduced with lentivirus expressing human TDP-43 and sequenced to compare exon skipping between mouse and human.(B) RNA-Seq analysis revealed numerous genes with skipped exons that are involved in a variety of molecular pathways related to intellectual disability, synaptic activity, and mitochondrial proteins (C).We identified exons with particularly high levels of exon skipping in the genes HYOU1, NUP93, and XPNPEP1 (arrows).(D-F) When cross-referenced with datasets from transgenic mice, we found that exons repressed in humans were not repressed in mice.Using RT-PCR, we validated skipping events in i3Neurons with primers located in exons adjacent to the repressed exon (G) or primers that spanned a skipped junction (H).(I) Analysis of UG repeats in human skiptic exons revealed that TDP-43's consensus motifs are found in both the repressed exon itself and adjacent intronic sequences.UG motifs appear slightly more frequently around the downstream 5' splice site, but with far shorter UG repeat lengths than those found adjacent to cryptic exons [73] Next, we wanted to explore whether TDP-43-mediated exon skipping represents a potential mechanism underlying the pathogenesis of ALS and TDP-43-related dementias.To study this, we designed single-band RT-PCR primers that could selectively amplify the unique junction produced by constitutive exon skipping.We profiled brain samples from FTD with TDP-43 inclusions, AD with or without TDP-43 inclusions, TDP-43 negative human brain controls, and i3Neurons.Previous studies have demonstrated that a cryptic exon found in the gene STMN2 is a robust biomarker for ALS-FTD [17][18][19]66].As expected, the STMN2 cryptic exon was detected in all four FTD samples and in 3 out of 4 AD TDP-43 positive samples and 2 out of 4 AD TDP-43 negative samples (Fig. 3B).AD cases without TDP-43 inclusions may still exhibit cryptic exons (STMN2) due to the observation of TDP-43 nuclear clearance without cytoplasmic aggregation [24,67,68].However, skipping events in at least three genes (HYOU1, NUP93, and XPNPEP1) could be detected in both disease cases and controls.
Since exon skipping was detected in some control cases, we explored whether aberrant exon skipping could be correlated with aging.To study this, we explored publicly available RNA-Seq archives using Snaptron and Recount3 [69][70][71][72], queried each skipped exon across thousands of brain samples available through the GTEx repository [73], and generally observed low levels of exon skipping, with slightly higher skipping frequencies in cerebellum tissue (Fig. 3C, Supplementary Fig. 7).Our data suggest that aberrant exon skipping, induced by excessive levels of nuclear TDP-43, is unlikely to play a significant role in disease pathogenesis.
Since aberrant exon skipping can occur independent from disease status, it will be important to avoid exon skipping when modeling TDP-43 proteinopathy.Overexpression of TDP-43 can be a valuable tool for investigating cytoplasmic toxicity, gain-of-function mechanisms, and post-translational modifications that may impact TDP-43 binding and other pathological interactions [74][75][76][77].Indeed, certain methods that induce cytoplasmic aggregation of TDP-43 can mimic loss-of-function [78,79].However, dose-dependent toxicity resulting from exon skipping may confound results when analyzing phenotypes from overexpression model systems, particularly since TDP-43 induced exon skipping appears to be highly species specific.Approximately 43% (30/69, Supplementary Table 1) of skipped exons are predicted to induce NMD, although skipped exons that retain reading frame are still predicted to impact protein structure (Fig. 3, Supplementary Fig. 8).Using the AlphaFold 2 protein prediction artificial intelligence system [80], we investigated the consequences on protein structure due to exon skipping and found significant impacts on protein structure and folding.For example, the XPNPEP1 isoform that results from exon skipping leads to complete disruption of the second domain of the protein (Fig. 3D,  H).For HYOU1, we observe a loss of alpha helical domains in the C-terminal region of the protein (Fig. 3G,  K).In NUP93, we predict that exon skipping would lead to dramatic changes in the C-terminal region that is crucial for nuclear pore complex assembly (Fig. 3E, I).These results suggest that inframe exon skipping can still dramatically affect protein structure and function, thereby leading to cellular toxicity.
A promising alternative to overexpression of wildtype TDP-43 (TDP-43 WT ) that could avoid exon skipping is the overexpression of TDP-43 NLSm , where the nuclear localization signal is mutated [50,52,58].Transgenic mice (rNLS8) have been generated where expression of TDP-43 NLSm can be induced by doxycycline (Dox) under the control of a neuron-specific driver line, the human NEFH-tTA promoter [53].However, since TDP-43 NLSm can enter the nucleus passively [54,81], we wanted to determine the maximum level of TDP-43 NLSm expression at which exon skipping can be detected.For this we used previously established doxycycline (Dox) inducible HEK-293 stable cell lines [58] to conditionally express TDP-43 WT (iGFP-WT) or TDP-43 NLSm (iGFP-NLSm) (Fig. 4A).Using RT-PCR, we found that exon skipping was present following doxycycline-dependent induction of either TDP43 WT or TDP43 NLSm Dox induction.However, the percentage of exon skipping for iGFP-NLSm induction reached only approximately 9% while iGFP-WT induction led to exon skipping of ∼ 90% (Fig. 4B).As previously described, induction of either iGFP-WT or iGFP-NLSm causes autoregulation of endogenous (See figure on previous page.)Fig. 3 TDP-43 exon skipping events are found in aging human brains but do not correlate with disease.(A) Alignment of syntenic mouse (mm10) and human (hg38) genomic sequences surrounding exons repressed by TDP-43 overexpression in human cells.Constitutively spliced exons in the genes XPNPEP1, NUP93, MYBBP1A, and HYOU1 are skipped in human cells but not mouse cells when TDP-43 is overexpressed.By contrast, the exon in DDI2 is repressed in both mouse and human cells.UG motifs (yellow highlights) are slightly enriched around the 3' and 5' splice sites (Supplementary Fig. 5).(B) We performed RT-PCR to amplify cryptic junctions or exon-repressed junctions in human brain samples from patients with AD pathology with or without TDP-43 inclusions, frontotemporal dementia with inclusions, and control patients who did not have TDP-43 inclusions and profiled the STMN2 cryptic exon [16,17].RT-PCR analysis showed that exon skipping occurred in both control and disease samples.(C) Exploration of skipping events through normal human aging was performed by analyzing RNA-Seq datasets from the Genotype-Tissue Expression (GTEx) project.PSI values of skiptic exons across human brain regions are shown for the age range of 60 to 69 years old (all age ranges are available in Supplementary Fig. 7).Exon skipping is found at low levels in most of the different brain areas analyzed, with slightly higher levels in the cerebellum.TDP-43, but also increases the total amount of TDP-43 protein (Fig. 4C-D).We estimate that a 50% increase in total TDP-43 compared to its endogenous level is enough to induce at least 40% of exon skipping.By comparison, a 100% increase in total TDP-43, due to iGFP-NLSm induction, leads to only 9% exon skipping (Fig. 4E).If we assume that 45% of iGFP-NLSm can passively diffuse into the nucleus (Table 1), we can estimate the total TDP-43 with the following calculation for Fig. 4D and E In animal models, first month of doxycycline induction results in overexpression of about 8 to 10-fold total TDP-43 (TDP-43 NLSm + endogenous TDP-43), suggesting that excessive levels of TDP-43 NLSm may induce higher levels of exon skipping [58].To test whether higher levels of TDP-43 NLSm overexpression promotes higher passive diffusion into the nucleus, we transfected HEK-293 cells with TDP-43 WT -P2A-GFP or TDP-43 NLSm -P2A-GFP and used FACS to obtain seven population fractions based on GFP intensity (Fig. 4F).We performed nuclei isolation on each fraction and quantified TDP-43 levels by immunoblotting and found that endogenous TDP-43 (TDP-43 end ) was eliminated by autoregulation from TDP-43 WT transfection in FACS populations with higher GFP intensity.This autoregulation was less evident in TDP-43 NLSm , as nuclear TDP-43 NLSm did not reach levels that were sufficient to completely repress TDP-43 end even in the highest GFP intensity population.By comparing the ratio of TDP-43 WT /TDP-43 NLSm at equivalent FACS populations, we estimated that 10-45% of TDP-43 NLSm can enter the nucleus.The wide range in diffusion percentages reflects the non-linear increase in TDP-43 WT transfection, as total TDP-43 levels begin to saturate and plateau despite an increase in GFP fluorescence (Fig. 4G).To estimate the minimal level of TDP-43 required to induce exon skipping, we also isolated RNA from each of the seven FACS populations isolated by GFP intensity and evaluated exon skipping using 2-band RT-PCR (Fig. 4H).We found that TDP-43 WT overexpression induced 90% exon skipping while TDP-43 NLSm saturated at 40% exon skipping (Fig. 4H).Since TDP-43 NLSm induces exon skipping in FACS fraction six at equivalent levels as the iGFP-NLSm Dox inducible system, i.e. both induce 9% exon skipping, we can use immunoblot analysis of isolated nuclei to estimate that nuclear TDP-43 NLSm is approximately 40% of nuclear TDP-43 WT (Fig. 4G).With these estimates, we extrapolated that total TDP-43 levels 1.1 to 1.5-fold above normal can begin to induce exon skipping.Together this suggests that cell line and animal models using TDP-43 NLSm could cause exon skipping toxicity if expression reaches sufficiently high levels.Estimations on the levels of TDP-43 required to induce exon skipping and the diffusion of TDP-43 NLSm are summarized in Table 1.

Discussion
In this study, we explore the molecular mechanisms by which excessive levels of TDP-43 in the nucleus can induce cellular toxicity.Our findings reveal that TDP-43 overexpression leads to skipping of constitutive exons that are normally incorporated into mRNAs under steady state conditions.This aberrant exon skipping appears to be driven by high concentrations of nuclear TDP-43 binding to suboptimal UG motifs located near these constitutive exons, although further studies are necessary to understand the exact mechanism of splicing repression.
Indeed, we observe that nearly all exons skipped due to TDP-43 overexpression are species-specific, at least when comparing between TDP-43 overexpression in mouse and human neurons.It is important to note that there is Table 1 Estimates for the amount of excess nuclear TDP-43 that is required to induce exon skipping across different experimental conditions.Our results suggest that an increase in TDP-43 that is greater than 1.1 to 1.5 fold over normal protein levels may lead to exon skipping toxicity.Models for TDP-43 overexpression should avoid increasing nuclear TDP-43 and aim to strictly limit excess TDP-43 to only the cytoplasm no clear consensus on the precise definitions and criteria used to classify constitutive, cryptic, and skiptic exons within the TDP-43 field.In this study, we consider exons with high percent spliced-in (PSI) values (> 90%) under steady-state conditions as constitutive, acknowledging that this threshold may differ from prior studies, and define skiptic exons as exons with high PSI values that are aberrantly skipped due to TDP-43 gain-of-function.We recognize that some identified skiptic events may represent increased alternative splicing rather than skipping of strictly constitutive exons, but further community effort is needed to precisely define these categories.Previous work has identified constitutive exon skipping (skiptic exons) in a knock-in mouse model that carries a TDP-43 mutation in its low complexity domain (Tardbp Q331K ) and suggested that skiptic splicing is a gain-of-function directly associated with disease-associated mutations [56].Our data suggests that the observed increase in total TDP-43 in heterozygous and homozygous Tardbp Q331K mice likely mediates skiptic splicing.By contrast, some disease-associated TDP-43 mutations appear to cause loss-of-function that can result in the incorporation of cryptic exons [82][83][84].It remains to be seen whether other mutations in TDP-43's C-terminal domain can also increase total TDP-43 levels and lead to constitutive exon skipping.In a limited comparison of PSI values between the TDP-43 WT and TDP-43 G298S transgenic models of this study, we do not detect skiptic events that are unique to TDP-43 G298S (Supplementary Fig. 9), but this does not rule out the possibility that other variants having mutation-specific effects on splicing [17,55] (Supplementary Data Files 3-5).Knock-in models of TDP-43 mutations may recapitulate potential shared mechanisms of disease between mouse and human, but our study suggests that skiptic exons and affected cellular pathways will be largely species specific.
Furthermore, while skiptic splicing could be detected in some human brain samples, these aberrant splicing events could also be detected in non-disease controls.By contrast, cryptic exons were exclusively detected in human disease samples, indicating their potential significance in disease pathogenesis.These findings have Fig. 5 Summary Diagram.TDP-43 is a highly autoregulated protein due to different forms of cellular toxicity when TDP-43 protein levels are either too low (cryptic exon incorporation) or too high (constitutive exon skipping, i.e. skiptic exons).Our study has demonstrated that these splicing deficits are linked specifically to nuclear TDP-43, whereas toxicity due to cytoplasmic TDP-43 remains to be fully elucidated.Future studies that avoid constitutive exon skipping (skiptic exons) may identify biomarkers for cytoplasmic-specific TDP-43 toxicity important implications for the interpretation and use of TDP-43 overexpression models in neurodegenerative disease research.
Our study underscores the need for caution when interpreting data obtained from TDP-43 overexpression models.We believe that TDP-43 induced exon skipping can be avoided when generating such models by ensuring that overexpressed TDP-43 is restricted to the cytoplasm and limiting nuclear TDP-43 protein levels to 130-150% above steady state.Cytoplasmic gain of function and nuclear loss of function of TDP-43 may both contribute to the pathogenesis of neurodegenerative diseases, but skiptic splicing may confound the interpretation of some model systems.For example, we observe minimal exon skipping in cell lines that stably express ΔNLS-TDP-43, but rNLS8 mice exhibit levels of nuclear TDP-43 that may be sufficient for skiptic splicing.Likewise, it remains to be determined whether AAV-mediated delivery of TDP-43 can also induce exon skipping, or whether TDP-43 autoregulation can minimize excessive nuclear TDP-43.
A notable limitation of our study is the lack of direct evidence connecting exon skipping with cell survival and disease pathology in vivo.We describe a potential pathogenic mechanism by which overexpression of various forms of TDP-43 (wildtype, mutant, or ΔNLS) induce aberrant exon skipping, but the context in which these exon skipping events occurs has not been fully addressed.TDP-43 overexpression may have other unexplored offtarget effects, with exon skipping perhaps representing only one part of a broader impact.Further research is needed to assess the consequences of exon skipping on cell viability and disease progression, and to explore the full range of TDP-43's effect in the cytoplasm.For example, TDP-43 overexpression has recently been linked to the misregulation and accumulation of NPTX2 [85].Although significant reductions in skiptic exons were not observed, exon skipping was nevertheless detected in bulk sequencing data at a range of _% to _% upon induction with TDP-43 (Supplementary Fig. 10), suggesting the possibility that a subset of cells may exhibit higher levels of exon skipping.
In conclusion, our study provides new insights into the complex molecular mechanisms underlying TDP-43 gain-and loss-of-function models.Our findings suggest that TDP-43 autoregulation is a highly conserved mechanism because both reduction and increases in TDP-43 protein levels lead to toxicity; depletion of nuclear TDP-43 leads to cryptic exon incorporation, while excess nuclear TDP-43 leads to constitutive exon skipping (Fig. 5).Future models of TDP-43 pathology should consider both cryptic exon inclusion and constitutive exon skipping to capture the complex role of TDP-43 in neurodegenerative diseases.

Ethics declarations
The authors declare no competing interests.

Antibodies Transgenic mouse generation
A Thy1.2 expression cassette on a pUC18 backbone was used to generate transgenic mice.Plasmids pTSC-TDP-WT and pTSC-TDP-G298S were constructed and submitted to the Transgenic Core Laboratory at the Johns Hopkins University School of Medicine for pronuclear injection using the hybrid mouse strain C57B6;SJL.Potential founders were screened by tail cutting, genomic DNA extraction, and PCR.The WT and G298S lines were maintained in the hybrid background and phenotypic characterization was started at the F3 generation.All mouse experiments were approved by the Johns Hopkins University Animal Care and Use Committee.

Hanging wire tests
F3 generation was followed to the end-stage (death or inability to right from a lateral decubitus position), and survival data was recorded.A subset of this population was selected for monthly hanging wire tests.Briefly, a mouse was placed on a wire grid, and the grid was shaken to encourage the mouse to grip the wires.The grid was then turned upside down and held level while the time for the mouse to fall was recorded, the endpoint was 60 s.Each mouse was tested 3 times, with the maximum hang time recorded as the result.

Tissue preparation for sectioning
Mice were anesthetized with an intraperitoneal injection of 15% chloral hydrate and anesthesia was monitored with limb and corneal reflex checks.Portions of quadriceps muscles were snap-frozen for sectioning.Mice were transcardially perfused with 50-100 mL of cold PBS, and then fixed on 4% PFA.The L3 and L4 dorsal root ganglia and attached dorsal and ventral roots were identified, removed, placed in a fixation buffer with 2% glutaraldehyde overnight, washed in PBS and embedded in Epon for EM.The brain and spinal cord were dissected and separated into right/left halves.One half of each was placed in a fixation buffer overnight at 4 °C with gentle agitation, and then held in PBS prior to embedding in paraffin and sectioning.The other half brain was half spinal cord was placed in a fixation buffer for 2 h, and then switched to sterile PBS with 30% sucrose overnight at 4 °C to be embedded in O.C.T and frozen in isopentane for cryostat sectioning.

Immunofluorescence and immunohistochemistry
Paraffin-embedded brain samples were sectioned in the sagittal axis and spinal cord samples were sectioned in the cross-sectional axis onto slides.Slides used for immunohistochemistry were incubated at 60 °C for 30 min, then deparaffinized in xylene and ethanol.Antigen retrieval was accomplished by incubating slides for 5 min in boiling 10mM sodium citrate buffer (pH 6.0).Slides were washed with PBS and blocked and stained using appropriate primary antibodies and reagents from the Vectastain Elite ABC kit (Vector Labs).Diaminobenzidine exposure was titrated to optimal contrast, sections were counterstained with Mayer's hemalaun, and then slides were dehydrated using ethanol and xylene and mounted.
Frozen gastrocnemius muscle was cut into 40 μm longitudinal sections using a freezing sliding microtome (Leica).Sections were separated into wells on a 12-well plate and blocked in IF blocking buffer: PBS with 5% normal goat serum and 0.5% Triton-X, slides were incubated overnight at 4 °C on primary antibody (rabbit anti-Synaptophysin and mouse anti-SMI-312), diluted in blocking solution.Sections were then washed with PBS with 0.5% Triton-X, incubated with α-bungarotoxin and goat antirabbit and anti-mouse secondary antibodies conjugated to Alexa Fluor-488 (Invitrogen), and washed again.Sections were spread on slides and coverslips were attached using Prolong Gold Antifade Reagent (Life Technologies).Slides were examined using a Zeiss LSM 510 Meta confocal microscope.

Muscle histology
Frozen quadriceps muscle embedded in O.C.T. were cut into 10 μm cross-sections onto slides using a cryostat (Leica).For H&E staining of muscle, sections were covered for 20 min with a fixation buffer (4% PFA in PBS).Sections were rinsed with distilled water, stained with Mayer's Hemalaun, rinsed, and stained with Eosin.After a final rinse, samples were dehydrated in ethanol and xylene, and coverslips were applied.For esterase staining, 25% α-naphthyl acetate, 5% acetone, 0.1% Pararosaniline HCL, and 0.1% Sodium Nitrate in 0.2 M Sodium Phosphate solution was used.The solution was applied to quadriceps sections for 5 min, rinsed in running tap water for several minutes and slides were then dehydrated in ethanol and xylene, mounted.Esterase and H&E-stained sections were analyzed under light microscopy.

I3neurons TDP-43 lentivirus transduction
i3Neurons [86] were transduced with lentivirus containing N-terminal Flag-tagged wild type TDP-43 at 1MOI, 2MOI, and 4MOI, respectively, at Brain physiological stage day 11.Neurons were harvested on Day 14 and dried ice frozen.Total RNA was isolated using Trizol extraction and used for downstream RNA sequencing analyses.

Immunoblotting
Tissues and cells were digested in RIPA Buffer with 1%, protease inhibitor (Roche Complete ULTRA mini tablet + EDTA), and phosphatase inhibitor (Roche PhosStop).Samples were centrifuged, and supernatants were saved.Protein concentrations in the supernatants were determined using a BCA assay (Pierce), and 20 µg of total protein was loaded into each well of a 10-20% Tris-Glycine gel or a 4-12% Bis-Tris gel (Invitrogen).Protein was transferred to a PVDF membrane (Millipore), blocked with 5% BSA in TBS with 0.1% Tween-20.Membranes were incubated overnight at 4 °C with primary antibody diluted in blocking buffer, then washed three times with TBS with 0.1% Tween-20, and incubated for 2 h with secondary antibody (Goat anti-mouse IgG-HRP or Goat anti-rabbit IgG-HRP, Sigma) diluted in blocking buffer.Three more washes with TBS with 0.1% Tween-20 followed, and then membranes were soaked in ECL solution (EMD Millipore Immobilon), dried, imaged on a Biorad image.Densitometric analysis was performed using Quantity One software (Bio Rad).

RNA extraction, library preparation, and RNA sequencing
Two ventral halves of spinal cords from TDP-43 WT and TDP-43 G298S transgenic animals and one littermate control for each line were dissected and transferred immediately into RNAlater storage reagent (Life Technologies).Total RNA from spinal cord tissues was extracted using the RNeasy Mini Kit (Qiagen).Total RNA from human tissue, QBI-293 stable cell lines, and HEK-293 cells were extracted using Monarch Total RNA Miniprep Kit (New England BioLabs, T2010S).To prepare RNA-Seq libraries, the TruSeq Stranded Total RNA Library Prep Kit (Illumina) was employed.Subsequently, the sample libraries were sequenced on an Illumina HiSeq for spinal cord samples and NextSeq for cultured cells.The obtained data was transformed into FASTQ files after demultiplexing.

Mouse animals were genotyped using the following primers: 5'-C G G A A G A C G A T G G G A C G G TG, 5'-G C C A A A C C C C T T T G A A T G A C C A, and 5'-A A G A T G G C A C G G A A G T C T A A C C
A T G, was used to generate a 386 bp band (spanning TARDBP exons 2, 3, and 4) in transgenic mice only and a 241 bp internal control band in all mice.LunaScript RT SuperMix Kit was used for cDNA synthesis.The following primers were used for RT-PCR in mouse tissue: Ddi2-F1:

C T T G T T G A G T, Psmd14-R1: T T G G C T T G G A A C A C T G G A T C
A, product: 473 bp (Full) or 353 bp (Skip).The following primers were used for PCR in human cells: HYOU1-F2:

C T G C A C A T C C C T A C A A T G G C, STMN2-R1: C A C A A G C C G C A T T C A C A T T C A, product: 167 bp; HYOU1-F1: C C G T A T G C A C C A T T G T G A C C, HYOU1-S5: C T G C T C A A T C T C A T C C T G T G C, product: 290 bp; NUP93-S2: G G T C A T A T T G A T A G A G C T T T T G A T A T C A G G, NUP93-R1: T G T A C T G A C A G T G T G C C G A C, product: 304 bp; XPNPEP1-S3: G C C T G G A T T A C A C A G G G C T A T T T, XPNPEP1-R1: A T T C G G C T T C C A G A C C C A A G, product: 177 bp; MYBBP1A-S2: C T G C A G C T A A T T C T G G A T G A C A A G, MYBBP1A-R1: C G C A G A G C C T T C T C C T T C T G
, product: 320 bp.Differences in RT-PCR product sizes were resolved on 4% agarose gels.

Percent splice-in bioinformatic analysis
FASTQ files were aligned using STAR [87] and a filtered splice junction file was processed to calculate percent of splicing (PSI) values following methodology described in ASCOT [71,72].Briefly, FASTQ files were aligned to the mm10 (mouse) or hg38 (human) genomes to generate BAM files.BAM files were converted into bigWig files using Megadepth [88].GTF transcript annotations from GENCODE were used to calculate gene expression values and perform exon sequence analyses (GEN-CODE v45 was used for human, GENCODE M34 was used for mouse).We calculated the 5´PSI or 3´as the ratio between the number of mapped splice junctions from one exon to another over the total amount of splice junctions including that exon the 5´or 3´splicing site.PSI values between controls and TDP-43 overexpression were compared and visualized using UCSC genome browser [89].Skiptic exon coordinates and PSI values are recorded in Supplementary Table 1.Custom code used in this project is available on Github: https://github.com/rcarmen1/skipticexonscomparisons/tree/main.

Calculation of repeat frequency
Calculation of UG/GU repeat frequency around a +/-400 bp window surrounding skiptic exons in Supplementary Fig. 5 was done by (i) masking all "UG" and "GU" as "YY"; (ii) replacing all "A, " "C, " "T, " "G" as "N"; (iii) identifying all pentamer and longer repeat sequences allowing for a single N insertion (i.e., "YYYYY, " "YYYYYY, " "YYYYYYY, " "YNYYYY, " "YYNYYY, " "YYYNYY, " "YYYYNY, " …); (iv) assigning the "Y"s in sequences from step 3 a value of 1 and all other sequences a value of 0; (v) aligning all sequences to the 3′SS and 5′SS, applying a vertical summation, and then dividing by the total number of sequences to obtain the repeat frequency per nucleotide position upstream and downstream of the skiptic exon.

Analysis of exon skipping in GTEx human samples
Splice junctions from the GTEx archive were queried using the Snaptron Web Services Interface and only brain, nerve and pituitary samples were collected [69,73].PSI values for skipped junctions were calculated as previously mentioned.We used the normalized quantification of transcripts per million (TPM) from the GTEx consortium (GTEx_Analysis_2017-06-05_v8_RSEMv1.3.0) to correlate PSI values and TDP-43 expression level [73].Plots from different brain regions were made using RStudio.

Protein structure modeling
Wildtype mRNA sequences for XPNPEP1 (ENST00000502935.6),NUP93 (ENST00000308159.10),MYBBP1A (ENST00000254718.9) and HYOU1 (ENST00000617285.5),were obtained from the GEN-CODE database and protein sequences were identified by translating the mRNA sequences with or without the exon of interest [90].Wildtype structures for NUP93 and HYOU1were downloaded from the AlphaFold Protein Structure Database.All other structures were generated using the Alpha Fold Monomer v2.0 pipeline (version 2.2.0) from the amino acid sequences on the Rockfish cluster at Johns Hopkins University [80,91].PyMOL was used to visualize the structures (Schrödinger, LLC.The PyMOL Molecular Graphics System, Version 2.5.4,2015).

Statistical analysis
Mouse data was analyzed using Microsoft Excel and GraphPad Prism software, with P values calculated using unpaired Student's t tests.P values less than 0.05 were considered significant.All quantitative values were reported as mean ± standard deviation (SD).

Fig. 1
Fig. 1 TDP-43 overexpression in mice leads to skipping of constitutive exons.(A) Human TDP-43 (TDP-43WT ) and TDP-43 carrying a G298S mutation (TDP-43 G298S ) were expressed under the weak Thy1.2 promoter in mice.(B) TDP-43 levels in the spinal cord and cortex of transgenic mice were compared to control mice using immunoblotting.In both transgenic lines, TDP-43 protein levels were elevated at approximately 1.5x and 1.3x higher in the spinal cord and cortex, respectively (* p < 0,05, ***p < 0.001).(C) We measured the hanging time of transgenic mice compared to their littermate controls and found a reduction associated with age, indicative of a motor neuron deficit.(D) Both transgenic lines had shorter survival times compared to non-transgenic (NT) controls (NT vs. WT: p = 0.0005, NT vs. G298S: p = 0.0035), but no differences were found between the two transgenic lines (WT vs. G298S: p = 0.1260).(E-G) RNA-Seq analysis on isolated mouse spinal cords revealed several examples of exon skipping (arrows) in both transgenic lines.(H) We further validated these findings by transfecting mouse N2a cells with human TDP-43 and performing RT-PCR to test whether TDP-43 expression alone was sufficient to induce exon skipping
product: 586 bp (Full) or 499 bp (Skip).The following primers were used for RT-PCR in human cells and brain samples: STMN2-F1: